The voltage-gated potassium channel Kv1.3 is significantly up-regulated in activated effector memory (TEM) cells in humans (Wulff H et al., 2003; Beeton C et al., 2006). As a consequence, Kv1.3 blockers constitute valuable new therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis (MS) and rheumatoid arthritis (RA) (Beeton C et al., 2011; Chi V et al., 2012).
HsTX1 toxin is a 34-residue, C-terminally amidated peptide from the scorpion Heterometrus spinnifer, which is cross-linked by four disulphide bridges (Lebrun B et al., 1997). Its amino acid sequence (SEQ ID NO: 1) and the locations of the four disulphide bridges are shown in FIG. 1. The solution structures of the synthetic toxin (Savarin P et al., 1999) and a chimaera consisting of the N-terminal half of the closely-related scorpion toxin maurotoxin (MTX) and the C-terminal half of HsTX1 (Regaya I et al., 2004) were found to be very similar to the canonical fold adopted by related scorpion toxins that contain only three disulphides, which consists of an N-terminal helix structure connected to a C-terminal two-stranded antiparallel β-sheet.
HsTX1 is a potent blocker of potassium channels. For example, it has been found that the peptide inhibited rat Kv1.3 channels with an IC50 of ca 12 pM and that it did not compete with 125I-apamin for binding to rat brain synaptosomal membranes (Lebrun B et al., 1997), although it did compete efficiently with 125I-kaliotoxin for binding to voltage-gated K+ channels on the same preparation (IC50 ca 1 pM). It is thus, a more potent Kv1.3 channel blocker than MTX and far more specific (Lebrun B et al., 1997). Subsequent studies have confirmed the marked preference of HsTX1 for Kv1.3 over Kv1.1, Kv1.2 and KCa3.1 channels (Regaya I et al., 2004). This high selectivity for Kv1.3 makes HsTX1 a potentially attractive candidate for the treatment of autoimmune diseases, as it has been shown that blockade of this channel in self-reactive TEM cells is effective in preventing the tissue damage associated with these conditions (Wulff H et al., 2003; Beeton C et al., 2006; Beeton C et al., 2011). Indeed, a considerable effort has been devoted to developing Kv1.3-selective analogues of the sea anemone peptide ShK (designated ShK-K-amide) as therapeutics for multiple sclerosis (Chi V et al., 2012; Pennington M W et al., 2012; Pennington M W et al., 2009) and one of these analogues has recently entered phase 1 clinical trials.
In an effort to understand the molecular basis for the potency and selectivity of HsTX1 for Kv1.3, the present applicants recently undertook a computational study of its interaction with Kv1.1, 1.2 and 1.3. Accurate models of Kv1x.-HsTx1 complexes were created using docking and molecular dynamics simulations. For each complex, the binding free energy of HsTx1 was determined from the potential of mean force calculations, with good agreement being found between the computed and experimental binding free energies. Comparison of the binding modes of HsTx1 with Kv1.1 and Kv1.3 revealed that the lower affinity of HsTx1 for Kv1.1 is due to its inability to come close to the pore domain of the channel, which prevents the pore-inserting lysine residue from making proper contacts with the tyrosine carbonyls in the “selectivity filter” of the channel.
In work leading to the present invention, the present applicants utilised the abovementioned models to design an analogue of HsTX1, namely HxTX1[R14A], with even greater selectivity for Kv1.3 over Kv1.1 and other channels. While not wishing to be bound by theory, it was predicted that the R14A mutation would perturb the binding mode of HsTX1. However, since the change in binding free energy associated with this mutation may not be reliably calculated using path-independent methods such as free energy perturbation, another approach using umbrella sampling molecular dynamics (MD) simulations was performed to determine the binding free energies of the peptide and its analogue from potential of mean force (PMF) calculations. This PMF method predicted that the R14A mutation in HsTX1 will yield >2 kcal/mol gain for the Kv1.3/Kv1.1 selectivity free energy relative to the wild-type (WT) peptide. Functional assays have subsequently confirmed the predicted selectivity gain for HsTX1[R14A] and thereby indicate that this analogue may be suitable for the development of a therapeutic agent and method for the treatment of autoimmune diseases.